Enzymes serve as biological catalysts that participate in a critical task detoxifying reactive oxygen species and breaking down toxic pollutants. Microbial enzymes from microalgae, macroalgae and bacteria hold potential promise and significance for biodegradation applications. Enzymes such as lipases, alkane monooxygenases, esterases, and dehydrogenases are associated with driving crude oil degradation. This study examines the biochemical effects of petroleum hydrocarbons from crude oil, sludge from tank bottom, and Effluent Treatment Plant (ETP) hydrocarbon sludge on enzyme activities of algae species. These pollutants contain mostly aliphatic compounds like butane, propane, and aromatic compounds like benzene, cyclohexane, which are toxic and carcinogenic in nature. Algal cultures were exposed to these pollutants at a pre-determined 9 mg/mL minimal inhibition concentration for 28 days, and the activities of lipase, esterase, dehydrogenase, and catalase were evaluated. Esterase activity increased by 4.00%–56.00% in cultures incubated consisting treatment compared to controls, which are algae cultures without any treatment, while catalase activity remained unchanged. Dehydrogenase and lipase activities showed minor variations, with Euglena sp. displaying 65.00% more lipase activity in cultures having treatment. GC-FID investigation of crude oil, sludge from tank bottom, and Effluent Treatment Plant (ETP) hydrocarbon sludge revealed hydrocarbon compounds ranging from Carbon 1 to 30 with propane predominating more than 90% of Total petroleum hydrocarbons (TPH). TPH abatement was found out to be 99.99% TPH in crude oil on treatment with Euglena sp. and Chlamydomonas sp., and 72.00% degradation by Chlorella sp. In ETP sludge, 99.00% TPH degradation was observed across three algae species, while sludge treatment achieved 99.90% degradation with Chlamydomonas sp. and Chlorosarcinopsis sp. These obtained results clearly allude to these algae species being capable degraders of petroleum hydrocarbons illustrating GC-FID’s functioning in remediation processes as an analyzing tool.