Recently, Shirai and Watanabe Royal Society Open Science, 9(1), 211128 (2022) developed OBNIS (Open Biological Negative Image Set), a comprehensive database containing images (primarily animals but also fruits, mushrooms, and vegetables) that visually elicit disgust, fear, or neither. OBNIS was initially validated for a Japanese population. In this article, we validated the color version of OBNIS for a Portuguese population. In study 1, the methodology of the original article was used. This allowed direct comparisons between the Portuguese and Japanese populations. Aside from a few emotional classification mismatches between disgust, fear, or neither-related images, we found that arousal and valence relate distinctively in both populations. In contrast to the Japanese sample, the Portuguese reported increased arousal for more positive valenced stimuli, suggesting that OBNIS images elicit positive emotions in the Portuguese population. These results showed important cross-cultural differences regarding OBNIS. In study 2, a methodological change was introduced: instead of the three classification options used originally (fear, disgust, or neither), six basic emotions were used (fear, disgust, sadness, surprise, anger, happiness), and a "neither" option, to confirm whether some of the originally "neither-related" images are associated with positive emotions (happiness). Additionally, the low-order visual properties of images (luminosity, contrast, chromatic complexity, and spatial frequency distribution) were explored due to their important role in emotion-related research. A fourth image group associated with happiness was found in the Portuguese sample. Moreover, image groups present differences regarding the low-order visual characteristics, which are correlated with arousal and valence ratings, highlighting the importance of controlling such characteristics in emotion-related research.