Introduction Lyme neuroborreliosis (LNB) is a tick-borne infection caused by bacteria Borrelia burgdorferi sensu lato that accounts for 10-15% of all Lyme borreliosis cases in Europe. LNB can present with a variety of neurological manifestations. We aimed to describe the typical anamnestic, clinical and laboratory features of patients diagnosed with LNB and to describe the differences between paediatric and adult cases. Additionally, we assessed the factors associated with definite LNB. Methods We retrospectively evaluated data of patients with suspected LNB had undergone lumbar puncture and were admitted to the Infectious diseases department of University hospital Bratislava, Slovakia, between September 2019 and May 2022. Patients were divided into three categories according to the diagnostic criteria of European Federation of Neurological Societies: A) cases with definite LNB, B) cases with possible LNB, C) non-LNB controls. Results In total, 139 patients were included in the analysis. 32 individuals were classified as definite LNB, 23 as possible LNB and 84 as non-LNB controls. 55.5% were females and 35.3% were children aged<18 years. 56.3% of patients with definite LNB reported a history of tick bite, and 21.9% a history of erythema migrans (EM). Peripheral facial nerve palsy (PFNP) was the most common clinical symptom in patients with definite LNB (65.6%), followed by headache (50.0%), fever (21.9%) and radicular pain (18.8%). In a univariate and multivariable analyses neither history of tick bite nor history of EM were significantly associated with definite LNB. Factors independently associated with definite LNB in multivariable analyses were (i) age < 18 years (aOR 7.89, 95% CI 2.00-31.03, p < 0,003), (ii) female gender (OR 6.34; 95% CI 1.66-24.17, p < 0.007), and (iii) facial nerve palsy (OR 10.54; 95% CI 2.41-55.19, p < 0.002). Conclusion We found that peripheral facial nerve palsy is the strongest predictor of definite LNB, and that the children<18 years and females in our study were more likely to be diagnosed with LNB. Our study also suggests that anamnestic data on history of tick bite and EM contribute little to the diagnosis of LNB and that the examination of CSF is essential for the diagnosis of LNB.