The present study was intended to investigate the biodegradation of acephate in aqueous media in the presence and in the absence of metal ions [Fe(III) and Cu(II)], and humic acid (HA). Biodegradations were performed using PS-5 (PS-5) isolated from the heavy metal polluted site. Biodegradations were monitored by UV-Visible, FTIR, and electron spray ionization-mass spectrometry (ESI-MS) analyses. ESI-MS analysis revealed that PS-5 degraded acephate to two metabolites showing intense ions at mass-to-charge ratios (/) 62 and 97. The observed kinetic was the pseudo-first order, and half-life periods () were 2.79 d (of PS-5 + acephate), 3.45 d [of PS-5 + acephate + Fe(III)], 3.16 d [of PS-5 + acephate + Cu(II)], and 5.54 d (of PS-5 + acephate + HA). A significant decrease in degradation rate of acephate was noticed in the presence of HA, and the same was confirmed by UV-Visible and TGA analyses. Strong aggregation behavior of acephate with humic acid in aqueous media was the major cause behind the slow degradation rate of acephate . New results on acephate metabolism by strain PS-5 in the presence and in the absence of metal ions [Fe(III) and Cu(II)] and humic acid were obtained. Results confirmed that strain PS-5 was capable of mineralization of the acephate without formation of toxic metabolite methamidophos. More significantly, the strain PS-5 could be useful as potential biological agents in effective bioremediation campaign for multi-polluted environments.