The present investigation aims to develop simultaneous extraction and conversion of inedible Madhuca longifolia seed oil into biodiesel by one-step acid-catalyzed in situ transesterification/reactive extraction process. Six different types of pretreatment were used to assess maximum yield of biodiesel. The maximum yield of 96% biodiesel was acquired with ultrasonic pretreatment at 1% moisture content, 0.61 mm seed grain size, 55 °C temperature, 400 rpm stirring speed, 15 wt% catalyst (H₂SO₄) concentration, and with 1:35 seed oil to methanol ratio in a time period of 180 min. This reaction kinetics precedes first order also the finest value of rate constant and activation energy were calculated as 0.003 min⁻¹ and 14.840 kJ mol⁻¹. The thermodynamic energy properties ΔG, ΔH, and ΔS are computed as 96457.172 J/mol, 12121.812 J/mol K, and − 257.12 J/mol K correspondingly. The enumerated outcome illustrates a heat absorb non-spontaneous/endergonic and endothermal reaction. The result of proposed work unveils ultrasonic pretreatment escalates the biodiesel efficiency and reactive extraction exemplifies the clean, cost-effective single-step approach for production of biodiesel from non-edible sources.