Background/Objectives: Otitis media (OM) is a common pediatric condition that significantly impacts hearing, language development, and quality of life. Emerging evidence suggests that vitamin D plays a crucial role in immune regulation and that deficiency may predispose children to recurrent OM. This study aimed to evaluate whether low serum vitamin D levels are associated with increased incidence and severity of OM in children aged 2-7 years. Methods: A cross-sectional study was conducted at a pediatric otolaryngology clinic in northwest Romania between November 2023 and April 2024. A total of 118 children were enrolled, including 87 children with documented episodes of OM within the preceding six months and 31 quasi-controls without recent OM episodes. Participants were stratified into three age groups (2-3, 4-5, and 6-7 years) and classified into clinical subtypes of OM (Acute Suppurative Otitis Media, Serous Otitis Media, and Acute Congestive Otitis Media). Serum 25-hydroxyvitamin D levels were measured using a chemiluminescence immunoassay and categorized as deficient (<= 20 ng/mL), insufficient (21-29 ng/mL), or sufficient (>= 30 ng/mL). Statistical analyses included one-way ANOVA with post-hoc tests, chi-square tests, linear regression, logistic regression, and Poisson regression. Results: Children with OM exhibited significantly lower vitamin D levels compared to controls, with the most pronounced deficiency observed in the Acute Suppurative OM group. One-way ANOVA revealed significant differences among the groups (F(3,114) = 82.30, p < 0.001), and linear regression demonstrated a strong inverse correlation between vitamin D levels and the frequency of OM episodes (r = -0.793, adjusted R-2 = 0.63, p < 0.001). Logistic regression indicated that vitamin D insufficiency significantly increased the odds of OM (OR approximate to 120.74, p < 0.001), while Poisson regression showed incidence rate ratios of 13.62 for deficient and 10.47 for insufficient vitamin D status (p < 0.001). Conclusions: The findings indicate that low serum vitamin D levels are significantly associated with an increased risk and frequency of otitis media in preschool-aged children. These results support the role of vitamin D deficiency as an independent, modifiable risk factor for recurrent OM, suggesting that vitamin D screening and supplementation could be beneficial in reducing the clinical and economic burden of this condition.