Lichens containing unicellular cyanobionts have received scant attention regarding their nitrogen- (N2-) fixing capacities. Thyrea girardii (Lichinomycetes), containing the putative cyanobiont Chroococcidiopsis, is the only species in this group so far reported to fix N2. Nitrogenase activity in T. girardii was higher in the light than in the dark, counter to overwhelming evidence that free-living unicells fix N2 in the dark only, providing temporal separation from oxygenic photosynthesis in the light. The original focus of the current work was to better understand the strategies by which N2 fixation and photosynthesis might operate concurrently in species of Lichinella (Lichinellaceae) and Thyrea (Porocyphaceae) but this aim was frustrated by the infrequency with which nitrogenase activity was detected in the populations sampled. This problem itself became a principal topic of this report. Lichinella nigritella, Thyrea confusa and T. girardii were collected in Catalonia, Spain and subjected to measurements of nitrogenase activity (acetylene reduction) and net CO2 exchange. Of 91 specimens of the three species combined that were tested, 49 had zero nitrogenase activity and only 23 had activity ≥ 5 nmol C2H4 g−1 h−1 with no obvious trends among species. Nitrogenase activity was consistently greater in the light than in the dark during diel dark-light-dark transitions. Maximum rates of nitrogenase activity recorded for L. nigritella, T. confusa and T. girardii were 133.4, 28.7 and 47.0 nmol C2H4 g−1 h−1, respectively. All specimens tested were photosynthetically competent, although rates of net CO2 exchange were comparatively low (maximum rates in L. nigritella and T. girardii were 37 and 16%, respectively, of maximum rates in Peltigera membranacea), and there was no evidence that photosynthetic rates were lower in thalli with appreciable nitrogenase activity. 15N natural abundance values for L. nigritella and T. girardii (δ15N = −3.41 and −0.84 ‰, respectively) were in agreement with published values for lichens with cyanobacterial photobionts. The results are discussed in relation to possible explanations for the low frequency of nitrogenase activity in these species, its occurrence in the light, and potential confounding factors (the presence of epiphytic heterocytous cyanobacteria and N pollution). It is suggested that the capacity of unicellular cyanobacteria to rapidly switch between non-diazotrophic and diazotrophic states may be advantageous in lichens with unicellular cyanobionts in habitats where N supply is only occasionally limiting.