Per- and polyfluoroalkyl substances (PFASs) are a class of compounds that persist in the environment globally. Besides being transported to the soil and sediments, which act as their sinks, PFASs can be transferred to several species of higher organisms directly or via bacteria, eliciting a wide range of adverse effects. Caenorhabditis elegans has been widely used in toxicological studies and life science research owing to its numerous advantages over traditional vertebrate models; notably, C. elegans has 65% conserved human-disease-associated genes and does not require ethical approvals for experimental use. This review covers a range of topics, from reported accumulation characteristics and lethal concentrations of PFAS in C. elegans to the mechanisms underlying the toxicity of PFAS at different levels, including reproductive, developmental, cellular, neurologic, oxidative, metabolic, immune, and endocrine toxicities. Additionally, the toxicity levels of some PFAS substitutes are summarized. Lastly, we discuss the toxicological mechanisms of these PFAS substitutes and the importance and promising potential of nematodes as in vivo models for life science research, epidemiological studies (obesity, aging, and Alzheimer’s disease research), and toxicological investigations of PFASs and other emerging pollutants compared with other soil animals or model organisms.