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Third-generation biofuels are currently considered to be the most resourceful medium for generating bioenergy. In the present study, microalgal strains were isolated from soil samples collected in Pakistan and characterized by 18S rRNA sequencing. The strains were identified as green algae Gloeocystis sp. MFUM-4, Sphaerocystis sp. MFUM-34, and Dictyochloropsis sp. MFUM-35. They were further studied for their potential to produce popular biofuels such as biodiesel, bioethanol, and biohydrogen. Under the test conditions, Gloeocystis sp. MFUM-4 emerged as the most suitable candidate, amongst the three new isolates, for biofuel production with a biodiesel production potential of 33.3% (w/v). Eight different environmental conditions were also tested to identify the most suitable condition for biohydrogen and bioethanol production using the newly isolated strains. Under light but in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), Gloeocystis recorded the highest capacity to produce both biohydrogen and bioethanol compared with the other strains that were examined.