Halotolerant Bacillus species, like other bacteria, adapt to stress conditions by modifying their fatty acid profiles to maintain membrane integrity. In this study, the fatty acid profiles of Bacillus halotolerans were investigated under stress conditions induced by lead (0.1% Pb), glycerol (0.1%), sodium chloride (0.1% NaCl), silver (0.05 ppm), and iron (0.1 ppm) nanoparticles. Fourier Transform Infrared and Gas Chromatography/Mass Spectrometry were employed to characterize the metabolic responses and identify the produced fatty acids. Among the applied stress conditions, the lead containing environment exhibited the highest level of fatty acid production (71.5 mg/L) and diversity. Also, different kinds of fatty acids with different applications were detected like omega-3 (α-Linolenic acid and Eicosapentaenoic acid), omega-6 (γ-Linolenic acid and Linoleic acid), myristoleic acid, palmitic acid, stearic acid, cis-hexadecenoic, cis-octadecenoic acid, and so on. These compounds are applicable in the pharmaceutical, medical, cosmetic, and food industries, whereas other fatty acids produced, such as biodiesel and its derivatives, also serve a wide range of purposes.