Multifrequency electron paramagnetic resonance (EPR) spectroscopy is a powerful tool that allows us to probe molecular structure and magnetic interactions of paramagnetic systems. By expanding from traditional X-band EPR into higher microwave frequencies, the terms of the spin Hamiltonian can be resolved and distinguished. Many complex and important paramagnetic systems are found in biology. Molecules of interest include metalloenzymes and proteins with organic radicals. Three examples of such systems described herein are phycocyanobilin:ferredoxin oxidoreductase (PcyA), soluble guanylate cyclase (sGC), and photosystem II (PSII). PcyA is a bilin reductase that carries out a four-electron reduction of biliverdin IX[alpha] to the chromophore phycocyanobilin. The reduction occurs with no metal or organic cofactors, with a mechanism involving substrate radical intermediates. The small anisotropy in the g tensor of one of these radicals that is not observable at X-band frequencies is resolved at 130 GHz and above. The g values (g = [2.00359(5), 2.00341(5), 2.00218(5)]) are determined from 406 GHz EPR, and the anisotropy of the g tensor is determined by the analysis of single crystals of PcyA mutants reduced by dithionite. The g tensor characterization along with DFT calculations places constrains on the possible protonation states of the radical intermediate. A state with both carbonyl oxygens protonated is identified, consistent with proposed mechanisms. The nitrosyl heme sGC exhibits an EPR spectrum with a complex axial EPR spectrum at X-band frequencies. The heme-containing domain [beta]1 truncations have a spectrum that resembles the spectrum of the full length protein. The [beta]2 heme-domain construct, on the other hand, has a much more simplified rhombic spectrum. The ambiguity of the origin of spectral features is resolved by simultaneous simulation of [beta]1 and [beta]2 spectra of the sGC complexes with bound 14NO and 15NO at X-, Q-, and D-band frequencies. The EPR spectrum of sGC is determined to be the superposition of a rhombic signal that resembles the spectrum of [beta]2 (g[subscript A] = [2.1507, 2.0245, 2.0102]), and an nearly axial signal g[subscript B] = [2.0600, 2.0580, 2.0125]. This two-component simulation fits observed spectral features better than previous simulations had. The two g tensors are interpreted as representing different five-coordinate conformations, with g[subscript A] being related to a high-activity state. PSII is the first enzyme in the electron transport chain in the chloroplasts of plant cells and cyanobacteria. The oxygen-evolution center where water oxidation occurs is located in PSII, and is composed of four Mn atoms and a Ca. X-ray crystal structures and spectroscopic models are considered. Multifrequency EPR spectroscopy of the "multiline" signal and the tyrosine radical Y[subscript Ḋ] of PSII is discussed, including 130 GHz EPR of single crystals. Other tyrosine models are examined. ENDOR spectroscopy of the Mn-cluster provides further characterization of these paramagnetic species. As part of the EPR study of these enzyme systems at higher microwave frequencies, the design and construction of an pulse and CW EPR spectrometer is described. The 130 GHz EPR/ENDOR spectrometer is an integral part of the array of instrumentation at the CalEPR facility at UC Davis that provides access to a range of microwave frequencies for paramagnetic biomolecules. Continued development of the spectrometer capabilities in CW EPR and impedance-matched ENDOR will further expand the reach of the facility to comprehend the structure and dynamics of enzymes such as PSII and PcyA.