Bioaccumulation of six perfluoroalkyl substances (PFAS) was assessed using the marine echinoderm Holothuria tubulosa Gmelin, 1791. Batch experiments were conducted to establish the relationship between concentrations in water, sediment and biota over 197 days. The sample treatment for the determination of compounds involves steps of lyophilization, solvent extraction and clean-up of the extracts with dispersive sorbents. PFAS were then analysed by liquid chromatography–tandem mass spectrometry. During contaminant exposure, detectable levels of compounds were found in all samples collected. Mean concentrations of selected PFAS were higher in sediments than in water samples. This fact is explained by the strong adsorption of these compounds into sediments. Sediment-water distribution coefficients (log Kd) were in the range 0.11 (PFBuA) to 2.46 (PFOA). Beside this, PFAS accumulation was observed in Holothuria tubulosa organisms. The uptake of PFAS was very rapid, reaching the maximum between 22 and 38 days of assay. Bioaccumulation factors (mean log BAF: 1.16–4.39) and biota sediment accumulation factors (mean log BSAF: 1.37–2.89) indicated a high bioaccumulation potential for the target compounds. Both parameters increased with perfluoroalkyl chain length (R2 > 0.93; p < 0.05). In organ-specific distributions of PFAS, greater concentrations were found in intestine than in gonads. Also, male specimens showed higher concentration levels than female (student t-test: tcal = 2.788, ttab = 2.262; p < 0.05). These data provide a detailed accounting of PFAS fate and distribution in the marine environment highlighting accumulation at lower trophic levels, a potential source for contamination in higher organisms.