We investigate sectorial operators and semigroups acting on noncommutative Lp-spaces. We introduce new square functions in this context and study their connection with H[infinity] functional calculus, extending some famous work by Cowling, Doust, McIntoch and Yagi concerning commutative Lp-spaces. This requires natural variants of Rademacher sectoriality and the use of the matricial structure of noncommutative Lp-spaces. We mainly focus on noncommutative diffusion semigroups. We discuss several examples of such semigroups for which we establish bounded H[infinity] functional calculus and square function estimates. This includes semigroups generated by certain Hamiltonians or Schur multipliers, q-Ornstein-Uhlenbeck semigroups acting on the q-deformed von Neumann algebras of Bozejko-Speicher, and the noncommutative Poisson semigroup acting on the group von Neumann algebra of a free group.