PLANTAGO major has been used to treat various diseases since ancient times. The present study aimed to assess the seasonal and habitat-related variations in the secondary metabolite content and antimicrobial activity of P. major leaf extracts. Eight habitats were selected for sampling plants in four seasons. The phytochemical screening of the ethanolic and chloroform extracts of P. major leaves from different habitats in different seasons showed the presence of cardiac glycosides, flavonoids, and phenolics. The highest cardiac glycoside and total flavonoid contents were observed in urban habitats and cultivated crops during winter, whereas the highest total phenolic content was observed in fallow lands during summer. Compounds separated and identified using high-performance liquid chromatography included phenolics such as ellagic acid, catechol, resorcinol, gallic acid, and phloroglucinol and flavonoids such as apigenin, luteolin, chrysoeriol, rutin, quercetin, kaempferol, and avicularin. The methanolic extracts of P. major leaves showed higher antimicrobial activity than aqueous and ethanolic extracts; the methanolic extract of P. major leaves from canal banks showed the highest activity against P. aeruginosa, followed by that from orchards against S. aureus. The results suggest that P. major contains phenolics and flavonoids that have potential medicinal applications. Moreover, the antimicrobial activity of the methanolic extract differed with habitat. Therefore, the present study confirms previously reported findings and indicates that the phytochemical contents and antimicrobial activity of the plant extracts differed with environmental factors (habitat and season). In future studies, the phytochemicals with antimicrobial activity need to be extracted, separated, purified, identified, and tested as pure or mixed compounds.