The presence of various heavy metals may influence the transformation of many organics by anaerobes under anaerobic conditions. Sometimes, the coexistence of both heavy metals and volatile fatty acids (VFAs) may cause an inhibitory impact on microbial species involved in such biotransformation. These toxic mixtures create disturbances in the bioreactors by inhibiting the ability of the microorganism to degrade the waste. To keep the anaerobic process horizontal, it is essential to know the concentrations of the heavy metals and VFAs during the wastewater treatment operations, otherwise, it would result in halting various operations. Other methods are time-consuming. However, the Microtox analysis provides quick and reliable information about the toxic concentrations of the mixtures by calculating the IC50 15 min luminosity inhibition through Photobacterium phosphoreum T3S. The current research work aimed to assess the impacts of Pb, Cd, Cu, and VFAs on luminescent anaerobic microbial species P. phosphoreum under laboratory conditions. The individual and combined