Background: The goal of this research was to measure patient safety culture in all Slovenian acute general hospitals. Methods: The hospital survey on patient safety culture questionnaire developed by researchers at Westat was used. Prior to this study, the questionnaire was psychometrically tested in three pilot hospitals and the results showed no need to modify the original model for the Slovene setting. Responses were scored on the 5-level Likert scale. The questionnaire was distributed to all hospital staff. After the exclusion of 159 questionnaires, which did not fulfil the inclusion criteria, 2932 (48.5 %) questionnaires were analysed. Descriptive statistics, confirmatory factor analysis, reliability of the scales, and construct validity were computed. Pot each item the percentage of positive, neutral and negative responses were calculated. Areas for improvement were defined as those answered positively by 50 % or less of the respondents. With one-way analysis of variance a comparison of the means among different sizes of the hospitals was performed. Results: The confirmative factor analysis showed adequate fit for the model and the questionnaire reliability was also adequate. The average positive percentage for all 12 composites was 53 %. Composite scores of patient safety culture revealed that none reached the level that would establish safety strengths. Statistically significant differences of means for hospital size were found (d.f. 2, 337,006; p < 0,0005) but were practically unimportant. Conclusion: The survey showed overall low patient safety culture, however it has provided the first insight into patient safety culture in acute general hospitals in Slovenia with several opportunities for improvement.