Ballast water is a vector for the transfer of microorganisms between ecospheres that can subsequently have a negative impact on native species of aquatic fauna. In this study, we determined the microbiota and selected physicochemical properties of ballast water from long- and short-range ships entering a southern Baltic port within a large estuary in autumn and winter (Police, Poland). Microbiological tests of the ballast water samples were carried out according to ISO 6887-1, and physicochemical tests were performed according to standard methods. Low amounts of oxygen (1.6–3.10 mg/dm3 in autumn and 0.60–2.10 mg/dm3 in winter) were recorded in all ship ballast water samples, with pH (above 7.90) and PSU (above 1.20) were higher than in the port waters. Yeast, mold, Pseudomonas bacteria (including Pseudomonas fluorescens), and halophilic bacteria as well as lipolytic, amylolytic, and proteolytic bacteria were found in the ballast water samples. Heterotrophic bacteria and mold fungi (log. 2.45–3.26) dominated in the autumn period, while Pseudomonas bacteria (log. 3.32–4.40) dominated in the winter period. In addition, the ballast water samples taken during the autumn period were characterized by a statistically significantly higher (p < 0.1) abundance of microorganisms (log 1.97–2.55) than in the winter period (log 1.39–2.27).