Optical tweezers have been little used in experimental studies on filamentous fungi. We have built a simple, compact, easy-to-use, safe and robust optical tweezer system that can be used with brightfield, phase contrast, differential interference contrast and fluorescence optics on a standard research grade light microscope. We have used this optical tweezer system in a range of cell biology applications to trap and micromanipulate whole fungal cells, organelles within cells, and beads. We have demonstrated how optical tweezers can be used to: unambiguously determine whether hyphae are actively homing towards each other; move the Spitzenkörper and change the pattern of hyphal morphogenesis; make piconewton force measurements; mechanically stimulate hyphal tips; and deliver chemicals to localized regions of hyphae. Significant novel experimental findings from our study were that germ tubes generated significantly smaller growth forces than leading hyphae, and that both hyphal types exhibited growth responses to mechanical stimulation with optically trapped polystyrene beads. Germinated spores that had been optically trapped for 25min exhibited no deleterious effects with regard to conidial anastomosis tube growth, homing or fusion.