Southwestern region of Bangladesh is very rich in floral diversity, and their diversified uses. An extensive survey was conducted to investigate ethnobotanical applications of botanical species by the community of Khulna, Bangladesh. We focused on plants and community relationships, identify the most important species used, determine the relative importance of the species surveyed and calculated the Fidelity level (FI) and Cultural Significance Index (CSI) concerning individual species. In total, we have listed 136 species of 114 genera under 52 families, of which 32% (45 species) were used for folk medicine. Inheritance of traditional knowledge of medicinal plants was the primary source of knowledge acquisition through oral transmission over the generations. However, only 34% of the informants were traditional herbal practitioners. Most of the medicinal uses are primly associated with anti-inflammatory, anti-microbial, antiseptic, expectorant, antidote, fever reduction, and pain relief.