This study investigated the effect of pesticide chlorpyrifos (CP) on a freshwater cyanobacterium Chroococcus turgidus NTMS12. The changes in chlorophyll‐a, proline, superoxide dismutase (SOD), catalase (CAT) activities and fatty acid composition of the test organism were analysed. Organism was grown at 6, 9 and 12 mg l⁻¹ of CP, and based on the chlorophyll‐a content, 6 mg l⁻¹ of CP was found to be the tolerable concentration. Hence, 6 mg l⁻¹ of CP was taken to evaluate the concentration of proline and activities of SOD and CAT at 48‐h exposure. The changes in the fatty acid profile were analysed after 7 days of exposure. Upon pesticide exposure, increased concentration of proline and activities of SOD and CAT were found. Significant changes in fatty acid profile have also been observed. However, polyunsaturated fatty acid content was decreased in treated cultures when compared with the untreated control. Changes in biochemical activities indicate that cyanobacteria C. turgidus NTMS12 undergo adaptive changes against CP‐induced oxidative stress. SIGNIFICANCE AND IMPACT OF THE STUDY: The fish pathogenic bacteria of genus Aeromonas, Vibrio, Edwardsiella and Streptococcus frequently cause severe outbreaks of diseases in cultured fish, and the genus‐specific multiplex PCR assay developed in this study can detect the bacteria of the four genera when present in the samples either alone or mixed. The mPCR assay is expected to identify the causative agents more efficiently than uniplex PCR or species‐specific multiplex PCR for clinical diagnosis, resulting in the earlier implementation of control measures. This mPCR assay provides a rapid, specific and sensitive tool for the detection or identification of common fish pathogenic bacteria in aquaculture practice.