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Simple Summary: Human poisoning due to the unknowing consumption of toxic pufferfish could represent a risk for EU citizens, as species currently present in the Mediterranean Sea can be accidentally caught by fishermen. Thus, monitoring pufferfish in the Mediterranean area, as well as assessing their toxicity, is pivotal for proper risk management. In this study both these aspects were addressed by collecting 56 pufferfish specimens of the species Sphoeroides pachygaster from Italian waters (Straits of Sicily) and by analyzing samples (liver, intestine, gonads, muscle, skin) from 20 specimens for the presence of toxins. The contribution of local fishermen to specimen collection was fundamental, confirming the importance of involving citizens in the monitoring of changes in the marine environment. In fact, this is the second largest sampling of S. pachygaster in this area. The specimens analyzed for the presence of toxins were found to be negative. However, further studies should be conducted, especially in the light of the changing environmental scenario, which could influence the production of toxins. Pufferfish (Tetraodontidae) inhabiting the Mediterranean Sea may represent an emerging public health risk due to the possible accumulation of marine neurotoxins such as tetrodotoxin (TTXs) and saxitoxin (STXs) in their tissues. In this study, the presence of pufferfish species in the Strait of Sicily (Lampedusa Island, Italy) was investigated using a citizen science (CS) approach, involving local fishermen. Samples (liver, intestine, gonads, muscle, skin) from 20 specimens were sent to the National Reference Laboratory on Marine Biotoxins for TTXs detection using a validated HILIC-MS/MS method on fish tissue. The presence of STXs was also screened in part of the specimens. Overall, 56 specimens identified as Sphoeroides pachygaster (Müller &Troschel, 1848) were collected. Data on their total length, body weight, fishing method and catch area (with relative depth temperature and salinity) were analyzed and compared with the S. pachygaster records reported in literature which were updated to 2022. All the analysed tissues were found to be negative for both TTXs and STXs. CS played an essential role in monitoring potentially toxic marine species in this investigation. Outcomes from this study, which is the first investigating S. pachygaster toxicity in Italian waters, may provide useful data for the proper assessment of this emerging risk. [ABSTRACT FROM AUTHOR]