To comprehensively reveal the effects of endogenous heavy metals on the desorption and release of n-hexadecane in calcareous soils from a karst area, the present work conducted desorption kinetic experiments and column experiments. This study also focused on the relationship between heavy metals and n-hexadecane desorption under various environmental factors including pH, ionic strength (IS), and rhamnolipid (RL). Results of desorption kinetic experiments demonstrated that the higher desorption content of Pb in the solution with a lower pH level created additional absorption sites for n-hexadecane, thereby inhibiting the desorption of n-hexadecane in soil samples. The co-desorption of Pb and n-hexadecane was observed in the presence of IS/RL. Particularly, a significant positive coefficient between n-hexadecane desorption and IS was observed (r = 0.904, p < 0.01), and Pb desorption positively correlated to RL (r = 0.810, p < 0.01). In contrast, results of column experiments showed that neither Cd nor Pb were released from the soil column. The limited interaction between soil and solution in the column led to the negligible influence of Cd and Pb on the release of n-hexadecane. The increased release efficiency of n-hexadecane with lower pH, reduced IS, or higher RL concentration can be attributed to the soil properties such as ion enrichment, higher soil pH, and weaker mineral compositions. The findings provide valuable insights into petroleum hydrocarbons mobility in karst areas.