Background: The intensive and injudicious use of pesticides in agriculture has emerged as a major concern due to its detrimental impact on aquatic ecosystems. However, the specific impact of broad-spectrum fungicides, such as Thiophanate-methyl (TM), on cyanobacteria remains a subject of ongoing research and debate. Methods: In order to fill this knowledge gap, The present study aimed to comprehensively investigate the toxicological effects of TM (10–30 µg/L) on the growth, photosynthetic pigments, oxidative stress, and biochemical composition of the non-nitrogen-fixing cyanobacterium Arthrospira platensis. Results: Our findings unequivocally demonstrated that TM exposure significantly inhibited the growth of A. platensis. Moreover, the decrease in chlorophyll content indicated a pronounced negative impact on the photosynthetic system of A. platensis caused by TM exposure. Notably, TM induced oxidative stress in A. platensis, as substantiated by a significant increase in lipid peroxidation (MDA) within the culture. Furthermore, the intracellular generation of hydrogen peroxide (H{}_2O{}_2) exhibited a positive correlation with higher TM dosages, while the levels of vital antioxidant enzymes, such as catalase (CAT) and ascorbate peroxidase (APX), exhibited a discernible decrease. This suggests that TM compromises the antioxidant defense mechanisms of A. platensis. Additionally, TM was found to enhance the activity of a plethora of enzymes involved in the detoxification of pesticides, including peroxidase (POD) and glutathione-S-transferase (GST), thereby indicating a robust detoxification response by A. platensis. Interestingly, exposure to TM resulted in a general suppression of biocomponent production, such as total proteins and total carbohydrates, which exhibited a diminishing trend with increasing TM concentration. Conversely, the lipid content witnessed a significant increase, possibly as an adaptive response to TM-induced stress. Conclusions: These findings contribute to a deeper understanding of the ecological implications of pesticide usage and emphasize the urgent need for the adoption of sustainable and environmentally-friendly agricultural practices to safeguard aquatic ecosystems.