Abstract Antimycin A (AA) is a widely used inhibitor to study photosynthesis and respiration. In photosynthesis, it is commonly used to inhibit a pathway of cyclic electron flow around photosystem I (CEF-PSI), but has also been reported to affect photosystem II (PSII), not involved in CEF-PSI. Although concerns have been raised about AA’s specificity, its impact on PSII activity remains unclear. AA3 was recently proposed as a more specific inhibitor of the same CEF-PSI pathway. In the mitochondrial respiratory chain, AA inhibits complex III, like myxothiazol. Here, we investigated the direct effects of AA, AA3, and myxothiazol on PSII activity and linear photosynthetic electron transport using isolated plant PSII and thylakoid membranes. AA, but neither AA3 nor myxothiazol, directly suppressed PSII activity and linear electron transport. Furthermore, the extent of AA’s effects was batch-dependent. Thus, we propose using AA3 to inhibit CEF-PSI, and myxothiazol to inhibit complex III, instead of AA.