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The struggling Spanish holm oak woodland situation associated with <i>Phytophthora</i> root rot has been studied for a long time. <i>Phytophthora cinnamomi</i> is considered the main, but not the only species responsible for the decline scenario. This study verifies the presence and/or detection of <i>Phytophthora</i> species in two holm oak areas of Spain (southwestern &#8220;dehesas&#8222; and northeastern woodland) using different isolation and detection approaches. Direct isolation and baiting methods in declining and non-declining holm oak trees revealed <i>Phytophthora cambivora</i>, <i>Phytophthora cinnamomi</i>, <i>Phytophthora gonapodyides</i>, <i>Phytophthora megasperma</i>, and <i>Phytophthora pseudocryptogea</i> in the dehesas, while in the northeastern woodland, no <i>Phytophthora</i> spp. were recovered. Statistical analyses indicated that there was not a significant relationship between the <i>Phytophthora</i> spp. isolation frequency and the disease expression of the holm oak stands in the dehesas. <i>Phytophthora quercina</i> and <i>P. cinnamomi</i> TaqMan real-time PCR probes showed that both <i>P. cinnamomi</i> and <i>P. quercina</i> are involved in the holm oak decline in Spain, but <i>P. quercina</i> was detected in a higher frequency than <i>P. cinnamomi</i> in both studied areas. Thus, this study demonstrates that molecular approaches complement direct isolation techniques in natural and seminatural ecosystem surveys to determine the presence and distribution of <i>Phytophthora</i> spp. This is the first report of <i>P. pseudocryptogea</i> in Europe and its role in the holm oak decline should be further studied.