Objectives A positive work environment (WE) is paramount for healthcare employees to provide good quality care. To stimulate a positive work environment, employees’ perceptions of the work environment need to be assessed. This study aimed to assess the reliability and validity of the Dutch version of the Culture of Care Barometer (CoCB-NL) survey in hospitals. Methods This longitudinal validation study explored content validity, structural validity, internal consistency, hypothesis testing for construct validity, and responsiveness. The study was conducted at seven departments in two Dutch university hospitals. The departments were included based on their managers’ motivation to better understand their employees’ perception of their WE. All employees of participating departments were invited to complete the survey (n = 1,730). Results The response rate was 63.2%. The content of the CoCB-NL was considered relevant and accessible by the respondents. Two factor models were found. First, confirmative factor analysis of the original four-factor structure showed an acceptable fit (X2 2006.49; df 399; p = <0.001; comparative fit index [CFI] 0.82; Tucker-Lewis index [TLI] 0.80; root mean square error of approximation [RMSEA] 0.09). Second, explanatory factor analysis revealed a five-factor model including ‘organizational support’, ‘leadership’, ‘collegiality and teamwork’, ‘relationship with manager’, and ‘employee influence and development’. This model was confirmed and showed a better fit (X2 1552.93; df 395; p = < 0.00; CFI 0.87; TLI 0.86; RMSEA 0.07). Twelve out of eighteen hypotheses were confirmed. Responsiveness was assumed between the measurements. Conclusions The CoCB-NL is a valid and reliable instrument for identifying areas needing improvement in the WE. Furthermore, the CoCB-NL appears to be responsive and therefore useful for longitudinal evaluations of healthcare employees’ work environments.