Although forest soils play an important role in the carbon cycle, the influence of topography has received little attention. Since the topographical gradient may affect CO2 emissions and C sequestration, the aims of the study were: (1) to identify the basic physicochemical and microbial parameters of the top, mid-slope, and bottom of a forest gully; (2) to carry out a quantitative assessment of CO2 emission from these soils incubated at different moisture conditions (9% and 12% v/v) and controlled temperature (25 °C); and (3) to evaluate the interdependence between the examined parameters. We analyzed the physicochemical (content of total N, organic C, pH, clay, silt, and sand) and microbial (enzymatic activity, basal respiration, and soil microbial biomass) parameters of the gully upper, mid-slope, and bottom soil. The Fourier Transformed Infrared spectroscopy (FTIR) method was used to measure CO2 emitted from soils. The position in the forest gully had a significant effect on all soil variables with the gully bottom having the highest pH, C, N concentration, microbial biomass, catalase activity, and CO2 emissions. The sand content decreased as follows: top > bottom > mid-slope and the upper area had significantly lower clay content. Dehydrogenase activity was the lowest in the mid-slope, probably due to the lower pH values. All samples showed higher CO2 emissions at higher moisture conditions, and this decreased as follows: bottom > top > mid-slope. There was a positive correlation between soil CO2 emissions and soil microbial biomass, pH, C, and N concentration, and a positive relationship with catalase activity, suggesting that the activity of aerobic microorganisms was the main driver of soil respiration. Whilst the general applicability of these results to other gully systems is uncertain, the identification of the slope-related movement of water and inorganic/organic materials as a significant driver of location-dependent differences in soil respiration, may result in some commonality in the changes observed across different gully systems.