Microphytobenthos (MPB) contributes up to half the primary production of estuaries. These microorganisms are sensitive to changes in sediment temperatures, particularly the extreme temperatures during exposure periods. This study investigates the physiological responses of MPB to freezing temperatures at two locations near Hobart, Tasmania during winter. Photosynthetic parameters were measured at 2 mm intervals to a depth of 10 mm. FV/FM values at three different distances from the shoreline at Kings Beach and Browns River in winter were between 0.584 and 0.617. rETRmax values were between 24.696 and 20.773. Maximum α values peaked in the subsurface rather than at the sediment surface. In vitro laboratory experiments (down to −5°C) showed little difference in response between the control and treatment groups, indicating no apparent effects of short term freezing on the MPB. Little change in photosynthetic parameters in response to freezing was probably associated with the resistance of light-harvesting reactions to freezing temperatures, recovery of the plasmalemma integrity or cryoprotection. Sediment composition and species composition were similar at both sampling sites. Therefore, responses of MPB were not due to species and grain size composition.