Step 1: Differentiate once.
\[
y = e^{4x}\cos 5x
\]
\[
\frac{dy}{dx} = e^{4x}(4\cos 5x - 5\sin 5x)
\]
Step 2: Differentiate again.
\[
\frac{d^2y}{dx^2}
= e^{4x}\big[(16-25)\cos 5x - 40\sin 5x\big]
\]
\[
= e^{4x}(-9\cos 5x - 40\sin 5x)
\]
Step 3: Substitute \( x = 0 \).
\[
\cos 0 = 1,\; \sin 0 = 0
\]
\[
\frac{d^2y}{dx^2}\Big|_{x=0} = -9
\]
Step 4: Conclusion.
\[
\boxed{-9}
\]