Objective To assess the values and practices that characterize the organizational culture of a private hospital in the state of São Paulo in the perspective of nursing professionals. Methods Quantitative, descriptive, cross-sectional study. Data collection was conducted between January and March 2013 using the Brazilian Instrument for Assessing Organizational Culture. Twenty-one nurses and sixty-two nursing aides and technicians participated in the study. The responses of the participants were coded into numerical categories, generating an electronic database to be analyzed by means of the software Statistical Package for the Social Sciences. Results Scores of cooperative professionalism values (3.24); hierarchical strictness values (2.83); individual professionalism values (2.69); well-being values (2.71); external integration practices (3.73); reward and training practices (2.56); and relationship promotion practices (2.83). Conclusion In the perception of workers, despite the existence of hierarchical strictness there is cooperation at work and the institution pursues customer satisfaction and good interpersonal relationships.