To investigate spatio-temporal responses of mega-benthos species to hypoxia, we conducted a bottom trawl survey with environmental observation in Mikawa Bay monthly from June to October. The number of deaths for each main mega-benthos species was estimated from standing stock in relation to fishing, recruitment, and growth. Further, natural mortality coefficient (M_t) and death ratio (Dn_ratio) under hypoxia expansion (i.e., June to September) were calculated. Distribution areas and standing stocks of each mega-benthos species decreased as hypoxia developed. Although M_t (month^-1) changed with the calculation period, the mean from June to September was estimated at 0.41–0.85, 0.42–0.46, 0.46–0.76 and 1.16–1.26 in flounder, swimming crab, mantis shrimp, and cockle, respectively. These values were higher than well-known natural mortality coefficients, which were calculated from biological parameters or observations conducted in normoxic regions. Calculated Dn_ratio was more than 50% in all analyzed species. The results of this study indicate that developed hypoxia causes mass mortality of main mega-benthos species. Moreover, expansion of hypoxic areas incidental to coastal development have potentially deteriorated the ecological corridor for the stone flounder and kuruma prawn populations, which use the inner area of the bay as a nursery.