Olive-mill wastewater (OMW) constitutes a major agricultural waste stream for which disposal is associated with significant environmental repercussions. No data are available on the effects of biotreated OMW and of the protective role of exogenously provided proline on plant physiology. In the present study, OMW was administered, either raw or previously treated by the white-rot fungus Pleurotus ostreatus, with or without proline amendment, to lettuce plants growing in sterilized sand. Biotreated OMW and proline addition resulted in significant moderation of OMW adverse effects on plant biomass production and ascorbic acid content, while their synergistic action alleviated the severe negative impact on net photosynthetic rate, water use efficiency and photosynthetic activity (Fv/Fo) invoked by the effluent. Moreover, biotreated OMW supplemented with proline, moderated the decrease in chlorophylls exerted by raw OMW, but it did not contribute at restoring carotenoids content. Restoration of plant transpiration was complete when biotreated OMW was used (with or without proline); proline alone mitigated the negative impact of OMW on photosynthetic efficiency (Fv/Fm and Fv'/Fm'). It seems that key photosynthetic parameters could be exploited as suitable evaluators of wastewater-induced plant toxicity, while plant fertigation with biotreated and/or supplemented OMW could be an interesting prospect in valorizing this effluent.