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The American horseshoe crab, Limulus polyphemus, has been reported to be illegally caught for use as Octopus spp. bait in some localities of the Yucatán Peninsula, Mexico. To learn if the octopus fishery could be negatively influencing the limulus populations in the area, L. polyphemus individuals were sampled twice a year, before and after the annual octopus fishing season, over 4 consecutive years (2015–2018) in Río Lagartos and San Felipe, both in the Ría Lagartos Biosphere Reserve (RLBR), Yucatán. Quadrants were drawn perpendicular to the mangrove edge, physicochemical parameters in the water (depth, temperature, dissolved oxygen, pH, salinity, and electrical conductivity) were determined, and granulometry of marine sediments was measured (Folk technique). The number of live specimens, number of females, and average length were recorded. No significant differences were found for the physicochemical properties of water or the recorded sedimentology (P > 0.05). Samplings revealed a low number of organisms per site: 22 in Río Lagartos and 24 in San Felipe, both from data before the start of the 2017 octopus fishing season. In the samplings after the start of the 2018 octopus fishing season, only 2 organisms were observed in Río Lagartos and none in San Felipe. The results showed a significant decrease in the number of live L. polyphemus specimens after the beginning of the octopus fishing season and a decrease in the average length of organisms, suggesting a negative effect of mollusk fishing activities on the horseshoe crab populations. More studies are required that integrate other physicochemical, biological, and socioeconomic factors to elucidate the potential threat of this fishery on L. polyphemus populations in the RLBR.