Nanoparticles have been reported to mitigate biotic and abiotic stresses in different fruits and vegetables. In this study, tomato fruit with black rot symptoms were collected and diagnosed. To control this disease, manganese oxide nanoparticles (MnO NPs) were synthesized in bacterial broth-culture. Based on microscopic, morphological, and genetic analyses, the pathogen causing black rot disease was identified as Aspergillus niger. MnO NPs were successfully synthesized in broth-culture of Bacillus subtilis, following the process of calcination and characterized. Fourier transform infrared (FTIR) spectrum revealed the existence of stabilizing and reducing agents (carboxylic acid, alkenes, and alkyl halides) on the surface of MnO NPs. X-Ray diffraction (XRD) analysis revealed the size (39 nm) and crystal-like nature of synthesized MnO NPs. Energy-dispersive X-ray spectroscopy (EDX) described the mass percentage of manganese (26.4%) and oxygen (23.3%). Scanning electron microscopy (SEM) displayed nearly spherical shape of MnO NPs and confirmed their nano-size. These MnO NPs exhibited significant mycelial growth inhibition of A. niger and notable control of tomato black rot disease of tomato. Though, all concentrations exhibited considerable effects, medium concentration of MnO NPs (2.5 mg/mL) performed best in both in vitro and in vivo analyses. At this concentration, tomato fruit maintained higher percentage of soluble solids, total sugars, reducing sugars and fruit firmness. These results proved very effective application of bacteria supplemented MnO NPs for the control of black rot disease of tomato. To our knowledge, this is the first study of tomato black rot, caused by A. niger in Pakistan.