Built during the 15th and 16th centuries, the Cathedral of St. James in Šibenik is considered one of the most important architectural works of Croatian cultural heritage and was included in the UNESCO list in 2000. To identify vulnerable points in the cathedral and enhance its resistance to future seismic effects, seismic ambient vibrations of the surrounding terrain and inside the cathedral were measured using the HVSR method. Based on 35 measurements in the area around the cathedral, microzonation of the ground frequency, H/V amplitude, depth of the base rock, and Vs30 values of the narrow area around the cathedral was performed. The preferred direction of ground movement was also determined. Microzonation revealed a significant change in soil properties in the southwest-northeast direction, i.e., from the coast to the interior. From 19 measurements at different points inside the cathedral, the frequencies of the dominant modes for each direction of oscillation were determined. The predominant direction of oscillation of the cathedral was established based on the amplitude of the H/V curves. By comparing measurements on the ground and within the building itself, critical structural elements were identified: the right side wall, the left gallery, and the dome. Certain structural units also exhibited diagonal modes of oscillation. This paper represents the first documentation describing the seismic response of the Cathedral of St. James concerning the local soil. It serves as a foundational work for further research and modeling of the response, with the ultimate goal of structural strengthening and the preservation of this valuable cultural heritage building.