Aims In order to efficiently control the corrosive sulphate-reducing bacteria (SRB), the main precursor of the microbial influenced corrosion (MIC) in oil industry, the ability of Trichoderma longibrachiatumDSMZ 16517 to synthesize silver nanoparticles (AgNPs) was investigated and their biocidal activity against halotolerant SRB was tested. Methods and Results The mycelial cell-free filtrate (MCFF) bioreduced the silver ions (Ag+) to their metallic nanoparticle state (Ag-0), which was presumptively indicated by the appearance of a dark brown suspension and confirmed by the characteristic absorbance of AgNPs at &(422nm). One-factor-at-a-time technique was used to optimize the effect of temperature, time, pH, fungal biomass and silver nitrate concentrations, stirring rates and dark effect. The dynamic light scattering (DLS) analysis revealed average AgNPs size and zeta potential values of 17 center dot 75 nm and -26 center dot 8 mV, respectively, indicating the stability of the prepared AgNPs. The X-ray diffraction (XRD) pattern assured the crystallinity of the mycosynthesized AgNPs, with an average size of 61 nm. The field emission scanning electron microscope (FESEM) and high-resolution transmission electron microscope (HRTEM) showed nonagglomerated spherical, triangular and cuboid AgNPs ranging from 5 to 11 +/- 0 center dot 5 nm. The Fourier transform infrared spectroscopy (FT-IR) analysis of the mycosynthesized AgNPs affirmed the role of MCFF as a reducing and capping agent. A preliminary suggested mechanism for mycosynthesis of AgNPs was elucidated. The mycosynthesized AgNPs expressed high biocidal activity against a halotolerant planktonic mixed culture of SRB. The HRTEM analysis showed a clear evidence of an alteration in cell morphology, a disruption of SRB cell membranes, a lysis in cell wall and a cytoplasmic extraction after treatment with AgNPs. This confirmed the bactericidal effect of the mycosynthesized AgNPs. Conclusion The biocidal activity of the mycosynthesized AgNPs against halotolerant planktonic SRB makes it an attractive option to control MIC in the petroleum industry. Significance and Impact of the Study This research provides a helpful insight into the development of a new mycosynthesized biocidal agent against the corrosive sulphate-reducing bacteria.