Biodiesel from crude palm oil (CPO) is conventionally produced via a two-step method: oil extraction followed by an esterification/transesterification reaction of oil to fatty acid methyl esters (FAME). In this study, a single-step in situ extraction and transesterification method (collectively known as reactive extraction) of CPO in palm fruit fiber from mesocarp to biodiesel was investigated. The reaction parameters studied include catalyst loadings (1–4% KOH w/v), reaction time (8–11 h) and mole ratios of methanol to oil (147:1–225:1). An optimum biodiesel yield of 97.25% w/w was obtained under the following reaction conditions: catalyst loading of 3.85% w/v of KOH, reaction period of 9 h and 36 min, methanol to oil mole ratio of 225:1 and reaction temperature of 60 °C. The biodiesel yield based on palm fresh fruit bunch (FFB) was found to be 272 and 175 g biodiesel per kg FFB by the single-step reactive extraction method and the conventional two-step extraction followed by reaction method, respectively