Background/Objectives: The ketogenic diet (KD) is a dietary model that can impact metabolic health and microbiota and has been widely discussed in recent years. This study aimed to evaluate the effects of a 6-week KD on biochemical parameters, gut microbiota, and fecal short-chain fatty acids (SCFAs) in women with overweight/obesity. Methods: Overall, 15 women aged 26-46 years were included in this study. Blood samples, fecal samples, and anthropometric measurements were evaluated at the beginning and end of this study. Results: After KD, the mean body mass index decreased from 29.81 ± 4.74 to 27.12 ± 4.23 kg/m2, and all decreases in anthropometric measurements were significant (p < 0.05). Fasting glucose, insulin, homeostasis model assessment of insulin resistance, hemoglobin A1C, urea, and creatinine levels decreased, whereas uric acid levels increased (p < 0.05). Furthermore, increased serum zonulin levels were noted (p = 0.001), whereas fecal butyrate, propionate, acetate, and total SCFA levels decreased (p < 0.05). When the changes in microbiota composition were examined, a decrease in beta diversity (p = 0.001) was observed. After the intervention, a statistically significant increase was noted in the Firmicutes/Bacteroidetes ratio (p = 0.001). Although Oscilibacter, Blautia, and Akkermensia relative abundances increased, Prevotella relative abundance and Bifidobacter abundance, which were the dominant genera before the KD, decreased. Moreover, the abundance of some pathogenic genera, including Escherichia, Klebsilella, and Listeria, increased. Conclusions: In healthy individuals, KD may cause significant changes in microbial composition, leading to dysbiosis and long-term adverse outcomes with changes in serum zonulin and fecal SCFA levels.