As alternatives to perfluorooctane sulfonate (PFOS) with shorter carbon chains or lower proportion of fluorine atoms, perfluorobutane sulfonate (PFBS), perfluorohexane sulfonate (PFHxS), and 6:2 fluorotelomer sulfonic acid (6:2FTSA) have been detected in various environmental media. However, it is unclear whether the toxicity of these alternatives is lower than that of PFOS. Therefore, this study investigated the toxicity and differences in PFBS, PFHxS, 6:2FTSA, and PFOS (0.2 mg/kg) after 56 d of exposure using the common invertebrate Eisenia fetida in soil as the test organism. The results showed that although PFOS, PFBS, PFHxS, and 6:2FTSA induced oxidative stress and apoptosis in earthworms and led to developmental and reproductive toxicity in terms of comprehensive toxicity, PFHxS > PFOS > PFBS >6:2FTSA. To reveal the mechanisms underlying the differences in toxicity between the alternatives and PFOS, we conducted molecular docking and transcriptomic analyses. The results indicated that, unlike PFOS, PFBS, and PFHxS, 6:2FTSA did not cause significant changes in antioxidant enzyme activity at the molecular level. Furthermore, PFOS exposure caused disorder in the nervous and metabolic systems of earthworms, and PFHxS disrupted energy balance and triggered inflammatory responses, which may be important reasons for the higher toxicity of these compounds. In contrast, exposure to 6:2FTSA did not result in adverse transcriptomic effects, suggesting that 6:2FTSA exerted the least molecular-scale toxicity in earthworms. The results of this study provide new insights into the environmental safety of using PFBS, PFHxS, and 6:2FTSA as alternatives to PFOS.